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Ann Thorac Surg 2003;76:2062-2070
© 2003 The Society of Thoracic Surgeons
a Department of Surgery, Division of Cardiovascular Surgery, Toronto General Research Institute, Toronto General Hospital, University of Toronto, Toronto, Ontario, Canada
Accepted for publication June 5, 2003.
* Address reprint requests to Dr Li, Toronto General Hospital, NUW G-108, 200 Elizabeth St, Toronto, ON M5G 2C4, Canada
e-mail: renkeli{at}uhnres.utoronto.ca
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Abstract |
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METHODS: A uniform transmural myocardial scar was created in adult rats by cryoinjury. Three weeks later the rats were randomly assigned to one of four blinded treatments: transplantation with 5 x 106 aortic smooth muscle cells (SMC, n = 12), ventricular heart cells (VHC, n = 13), skeletal muscle cells (SKC, n = 13) or culture medium alone (control, n = 11). The ACE inhibitor group (n = 8) received enalapril (1.0 mg/kg per day), also beginning 3 weeks after cryoinjury. Five and 12 weeks after transplantation, left ventricle (LV) function was assessed in a Langendorff apparatus, and histologic and immunohistological evaluation of the LV scars was performed.
RESULTS: At 5 weeks, greater scar elastin content and better LV function was noted with cell transplantation or ACE inhibitor therapy compared with control rats (p < 0.05). Twelve weeks after transplantation, cell-transplanted rats still had greater elastin content and better LV function than control rats, although elastin content and LV function had declined in ACE inhibitor-treated animals to levels below those observed in control rats (p < 0.05).
CONCLUSIONS: Transplantation of SMC, VHC, and SKC preserved ventricular function equivalent to the effects of an ACE inhibitor. Muscle cell transplantation, but not ACE inhibitor therapy, continues to be effective later after cryoinjury. No differences were detected between the muscle cells.
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Introduction |
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Previous studies have demonstrated that fetal cardiomyocyte transplantation can prevent heart failure in a variety of models of cardiac injury [4, 5]. We found that the fetal cardiomyocytes were eventually rejected despite cyclosporin therapy [6]. Therefore, we have investigated adult cells for autologous transplantation. Skeletal myoblasts have been used successfully in both animals [7–9] and in humans [3]. Both atrial [10] and ventricular [5] heart cells and smooth muscle cells [11] have been used successfully to prevent cardiac dilation and progressive heart failure. Both endothelial cells [12] and fibroblasts [13, 14] engraft in the injured myocardium inducing angiogenesis, but do not improve heart function. Therefore, we compared three muscle cells that are required to modify remodeling after cardiac injury and prevent cardiac dilatation and dysfunction.
The mechanism by which cell transplantation prevents heart failure remains obscure. Engrafted muscle cells have not been demonstrated to beat synchronously with the heart. The muscle cells alter the extracellular matrix and modify the elasticity of the injured region. Therefore, we evaluated the elastin content of the injured region compared with animals who received media injection without cells. To assess the magnitude of the benefit with muscle cell implantation, we also included a group who received the ACE inhibitor, enalapril.
Cell transplantation will likely require cryopreservation to facilitate clinical application. We have previously demonstrated that cryopreservation can preserve cells for subsequent transplantation [15]. However, the cryopreservation process limits cell proliferation and alters engraftment. Therefore, we used cryopreservation before isogenic cell transplantation to determine the feasibility of this technique for all three muscle cell types.
Cell transplantation is a complex process, which will require multiple investigations before clinical application. These initial studies are intended to initiate this process. Further study of the process of cryopreservation in each of these cell types will be required to make definitive statements about this preservation technique.
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Material and methods |
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Cell isolation and cryopreservation
Lewis rats underwent general anesthesia induced by intramuscular ketamine (22 mg/kg body weight) and intraperitoneal pentobarbital (30 mg/kg body weight). Left ventricular myocardium, aorta, and the vastus muscles of both thighs were harvested for isolation of heart cells, vascular smooth muscle cells, and skeletal muscle cells, respectively. Ventricular heart cells were isolated by digesting the minced myocardium in a solution of 0.2% trypsin and 0.1% collagenase, as described previously [5, 16, 17]. Vascular smooth muscle cells were isolated by protease digestion of the aorta after enzymatic removal of the endothelium [18]. These cells were then cultured in Iscove's modified Dulbecco's medium containing 10% fetal bovine serum (FBS), 0.1 mmol/L ß-mercaptoethanol, 100 IU/mL penicillin, and 100 µg/mL streptomycin (Gibco Laboratories, Life Technologies, Grand Island, NY). Skeletal muscle cells were isolated by mincing the vastus muscles into small pieces and digesting with 0.2% type 1 collagenase (223 U/mg) for 15 minutes, followed by 0.15% pronase (3.9 U/mg) for 15 minutes. The isolated cells were cultured on laminin-coated dishes in M199 medium containing 10% FBS and antibiotics.
When the cultured cells had reached 70% to 90% confluence at their second passage, they were detached from the culture dishes and resuspended at a concentration of 4 x 106 cells/mL before being mixed with an equal volume of cryopreservation solution (20% DMSO, 80% FBS). The cells were placed in cryopreservation containers (Cryovials, Nalgene, San Francisco, CA) at -80°C for 24 hours before storage in liquid nitrogen for 4 weeks.
The cryopreserved heart and smooth muscle cells were thawed in a 37°C water bath and seeded into T-175 flasks containing 45 mL of culture medium as above. When the cells had reached 80% confluence, they were detached and resuspended for transplantation. The skeletal muscle cells were seeded into 60-mm laminin-coated plates after thawing, and transplanted at 70% confluence, before aggregation into myotubes.
Immunocytochemistry for cell identification
Before transplantation, the cultured cells were stained with monoclonal antibodies against ß-myosin heavy chain for heart cells [5, 16, 17]; desmin and myogenin for skeletal muscle cells [8, 9]; and 
-actin for smooth muscle cells, as described previously [11, 18]. Antibodies against factor VIII were used to identify vascular endothelial cells [12]. Briefly, cultured cells were fixed with 100% methanol at -20°C for 20 minutes. Endogenous peroxidase was then blocked using 3% H2O2 for 10 minutes at 42°C and 2 N HCl for 30 minutes at 21°C. After rinsing with phosphate-buffered saline (PBS) 3 times, the cells were incubated with antibodies against ß-myosin heavy chain, desmin, myogenin, -smooth muscle actin, or factor VIII for 16 hours at 21°C. Negative control samples were incubated in PBS without the primary antibodies. After being rinsed three times for 15 minutes each, the cells were then incubated with goat anti-rabbit immunoglobulin G conjugated to peroxidase at 37°C for 45 minutes. The samples were washed three times for 15 minutes each with PBS, and then immersed in diaminobenzidine-H2O2 (2 mg/mL diaminobenzidine, 0.03% H2O2 in 0.02 mL/L phosphate buffer) solution for 15 minutes. After washing with PBS, the samples were covered with a crystal mount and photographed. Fields of five cultures (from 5 separate animals) were randomly selected and the numbers of positively and negatively stained cells were counted. The percentage of positively stained cells was expressed as mean ± standard deviation.
Cell labeling and preparation for transplantation
To identify the transplanted cells in the myocardial scar, 20% of the plates of cells were prelabeled with bromodeoxyuridine (BrdU, Sigma, Ontario, Canada) before transplantation [4, 5, 19]. In brief, 150 µL of 0.4% BrdU was added into each culture dish and incubated with the cells for 48 hours. The cells were then dissociated from the dishes and resuspended in serum free culture medium at a concentration of 5 x 106 cells in 100 µL for transplantation.
Myocardial scar generation and cell transplantation
Under general anesthesia as described previously, adult rats were intubated; positive pressure ventilation (180 mL/min) was maintained using a Harvard ventilator (model 683). The rat heart was exposed through a 2-cm left lateral thoracotomy. A transmural cryoinjury was produced by application of a cryoprobe cooled in liquid nitrogen to the left ventricular free wall for 1 minute, repeated 12 times [4, 11, 13]. The muscle and skin were then closed with 3-0 silk sutures. Penicillin G benzathine (150,000 U/mL) was given intramuscularly.
Three weeks after cryoinjury, the rats underwent general anesthesia and the heart was exposed through a median sternotomy. A pursestring suture of 6-0 Prolene (Ethicon Inc, Somerville, NJ) was placed around the intended site of injection in the myocardial scar, to prevent leakage of the injected cells. The rats were randomly assigned to one of five groups for different cell transplantation and at two times for evaluation: smooth muscle cells (n = 12, 6 at 5 weeks and 6 at 12 weeks), left ventricular heart cells (n = 13, 6 at 5 weeks and 7 at 12 weeks), skeletal muscle cells (n = 13, 6 at 5 weeks and 7 at 12 weeks), culture medium alone (control, n = 11, 5 at 5 weeks and 6 at 12 weeks), or no injection at all but subsequent administration of enalapril at 1.0 mg/kg per day (ACE inhibitor, n = 8, 4 at 5 weeks and 4 at 12 weeks). The personnel performing the transplantations and subsequent evaluations were blinded to the transplantation group in the four groups in which injections were given, as the cells had been cultured and prepared by separate personnel. In the cell-transplanted groups, 100 µL of the cell suspension, containing 5 x 106 cells, was injected using a tuberculin syringe into the center of the left ventricular free wall scar. The chest was closed with 3-0 silk sutures, and antibiotics administered as described previously.
Evaluation of ventricular function
Five or 12 weeks after transplantation, rats were anesthetized as described previously. The hearts were rapidly excised and function was evaluated in a Krebs-Henseleit buffer-perfused Langendorff preparation. A latex balloon was passed into the left ventricle through the mitral valve and connected to a pressure transducer (model p10EZ, Viggo-Spectramed, Oxnard, CA), a transducer amplifier, and a differentiator amplifier (model 11-G4113-01, Gould Instrument System Inc, Valley View, OH). After stabilization for 20 minutes, coronary flow in the empty beating state was measured in triplicate and the mean value recorded. The volume in the intraventricular balloon was increased by addition of water in 20-µL increments from 40 µL until the left ventricular end-diastolic pressure reached 30 mm Hg. Systolic, diastolic, and developed pressures were recorded and calculated as described previously [4, 10, 11, 12].
Morphology and histology
After evaluation of ventricular function, the hearts were arrested by intracoronary perfusion of 5 mL of 20% KCl solution. The passive diastolic pressures in the arrested hearts were recorded at different balloon volumes. The hearts were fixed in 10% formaldehyde and sectioned into 1-mm slices. The sample slices were sectioned for histology at 10-µmol/L thickness and stained with hematoxylin and eosin as described in the manufacturer's specifications (Sigma Diagnostics, St. Louis, MO). Sections were stained for ß-myosin heavy chain [4, 5, 16, 17] and -smooth muscle actin [11, 18] as described previously. Staining with monoclonal antibodies against BrdU was used to identify the transplanted cells [4, 5, 11–13, 19]. The characteristics of the extracellular matrix were evaluated by staining with Verhoeff's elastic trichrome technique, which stains nuclei and elastic fibers black, collagen green, and myocytes red [20]. After deparaffinization, the samples were stained with Verhoeff's stain solution [5% alcoholic hematoxylin:10% Ferric chloride:Verhoeff's iodine (iodine and potassium iodine in a 1:2 ratio) in a ratio of 5:2:2] for 20 minutes. The samples were then destained in a 2% ferric chloride solution until nuclei and elastic fibers could be clearly seen by light microscopy. Nonspecific background staining was removed with 5% sodium thiosulfate. The muscle cells were then stained with 1% Biebrich Scarlet in 1% acetic acid for 1 minute. Samples were then treated with a solution (5% phosphotungstic acid and 5% phosphomolybdic acid in a ratio of 1:1) for 1 minute. Collagen was stained last with 1% fast green in 1% acetic acid for 2 minutes and sections were rinsed briefly in 1% acetic acid before being dehydrated, mounted and photographed. Photographs were subjected to computerized image analysis to quantitate the percentage of black-stained elastin within each section.
Data analysis
All data are expressed as mean ± standard deviation. SAS software (SAS Institute, Cary, NC) was used for all analyses. Data on morphology, histology, and volumes were analyzed by analysis of variance. Data on ventricular function, including systolic, diastolic, and developed pressures were analyzed by analysis of covariance, using group and balloon volume as the covariates. Statistical significance was assumed at a p value of less than 0.05. When the F ratio associated with the analysis of variance or analysis of covariance was statistically significant (p < 0.05), then a post hoc evaluation was performed by Tukey's multiple range test to identify where the differences occurred.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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-smooth muscle actin (80% ± 3%, Figs 1E, 1F). None of the cultures was pure and antibodies against factor VIII demonstrated that endothelial cells comprised 26% ± 4% of the cells in the heart cell cultures, 11% ± 4% of the cells in the skeletal muscle cell cultures, and 14% ± 2% of the cells in the smooth muscle cell cultures. There was also a small number of cells resembling fibroblasts in all cultures.
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-smooth muscle actin (Figs 2E, 3E). In the ventricular heart cell-transplanted groups, ß-myosin heavy chain was noted in the engrafted region (Figs 2B, 3B). In the skeletal muscle cell-transplanted group, striated muscle was observed in the scar. No lymphocytic infiltrate was histologically apparent in any of these isotransplanted rat hearts.
Elastin in the transplanted scar
The composition of the extracellular matrix varied significantly between groups. In the control group, minimal elastin staining was noted at 5 and 12 weeks (Figs 4, 5).
In contrast, elastin staining was much more pronounced in hearts
transplanted with ventricular heart cells, smooth muscle cells, or skeletal muscle cells, at both 5 and 12 weeks. The ACE inhibitor-treated hearts demonstrated significant elastin staining 5 weeks after the initiation of therapy, but this finding did not persist. After 12 weeks of therapy, cardiac elastin staining appeared to be minimal, resembling that in the control hearts that did not receive ACE inhibitor.
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Passive ventricular volumes
Five weeks after cell transplantation, a significant difference was found between groups by analysis of covariance (p = 0.005). The multiple range t test (Tukey's test) identified smooth muscle cells as being statistically (p = 0.05) different from media injected control hearts (Fig 6A).
Twelve weeks after cell transplantation, both control and ACE inhibitor hearts dilated significantly, resulting in a significant group effect (p = 0.001) (Fig 6B). Both heart cells and smooth muscle cells were significantly (p = 0.01) different from either control or ACE inhibitor group hearts.
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0.001, Fig 7A).
Twelve weeks after cell transplantation, developed pressure deteriorated in both the media-injected control and the ACE inhibitor-treated hearts. The three cell-transplanted groups had higher developed pressures at similar balloon volumes than the media-injected control or the ACE inhibitor groups (ANOCOVA p = 0.001, Fig 7B) 12 weeks after cell transplantation.
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Animal model
Although the cryoinjury rat heart model used in this study is not clinically applicable, the injury forms a homogeneous scar that has with a distinct border with the host myocardium. Since the cryoinjury-derived scar is of constant size and location, the number of animals required to obtain a statistically significant power for statistical comparison is smaller than that required with the coronary ligation model [13]. The coronary ligation rat model is undoubtedly more clinically relevant. However, the wide range of infarct sizes with borderline viable cardiomyocytes and islands of viable cardiomyocytes within the scar necessitates the use of more animals for statistical comparisons. Both models have a similar postinjury inflammatory reaction and can result in a similar progressive ventricular dilation and congestive heart failure [4, 11, 12]. To assess clinical relevance of the results in this study, we believe that the results of both the cryoinjury and coronary ligation models in small animals need to be confirmed in a large animal coronary ligation model.
Cell type
Various cells used for transplantation, and in most studies they have engrafted in the heart. Skeletal myoblasts are currently undergoing clinical trials [2]. After implantation, these cells undergo phenotypic modulation [8]. Recent evidence suggests that the engrafted myoblasts orient in relation to the myocardium [21] and may contract [22]. However, the skeletal myoblasts do not form gap junctions and do not beat synchronously with the host myocardium. Smooth muscle cells proliferate in vivo and respond to hemodynamic stresses with hypertrophy and hyperplasia [11, 18]. Vascular smooth muscle cells can be obtained from peripheral veins and maintain their smooth muscle cell characteristics (-smooth muscle actin, Figs 2E, 3E) even after 12 weeks of implantation. Ventricular heart cells may be cardiomyofibroblasts, but also have some characteristics of cardiomyocytes. They maintain expression of ß-myosin heavy chain (Figs 2B, 3B). They express some muscle proteins and appear to orient when implanted into a left ventricular scar [5, 19]. They do not beat when engrafted in the heart.
All three adult muscle cells appear to modify ventricular remodeling, prevent cardiac dilatation, and preserve systolic function when implanted into an injured region. Further studies will be required to make a definitive comparison of alternative cell types in a more clinically relevant large animal model. This study suggests that all three cell types are feasible and are potential candidates for clinical application.
Magnitude of effect
We compared muscle cell transplantation with an ACE inhibitor to estimate the magnitude of the effect that can be anticipated with cell transplantation. We found that at 5 weeks after cell transplantation the magnitude of the effect was similar. However, 12 weeks after cryoinjury, hearts treated with enalapril dilated and were unable to generate adequate systolic pressures despite continued treatment. The lack of sustained efficacy for ACE inhibitor has been demonstrated previously [23] and may be related to the delayed initiation of therapy in our animals or reflect the large homogeneous scar formed by the cryoinjury. Twenty-five percent of the left ventricular mass was cryoinjury-derived scar. The dose used was similar to that shown to be beneficial by others [24], but may not be optimal. We would anticipate that combining ACE inhibitor and cell transplantation would have an additive effect. Myoblast transplantation has been demonstrated to preserve ventricular function for at least a year in a sheep model [25] and 2 years in humans [26].
Mechanism of benefit
Recent studies suggest that myoblasts may contract when engrafted in an infarcted region in a sheep model [22]. However, myoblasts do not communicate with the host myocardium and do not beat synchronously with the heat. Therefore, the implanted muscle cells did not contribute directly to contractility in the injured regions. We therefore investigated alternative mechanisms of benefit. Each of the cell types modified the surrounding matrix and preserved or produced elastin both in the center of the infarct (Fig 5A) and in the border surrounding the cryoinjury (Fig 5B). We found an increase in the elastin content in both regions with cell transplantation and early after treatment with ACE inhibitor. Therefore, we hypothesized that the mechanism of benefit of cell transplantation in this model relates to modification of the remodeling process with preservation of the elastic components of the myocardium. Cell transplantation may alter the matrix components of the injured heart region as well as the normal myocardium to prevent congestive heart failure [20]. Cell transplantation prevented the thinning and dilatation which constitutes remodeling after cryoinjury. Maintenance of the thickness and elasticity of the ventricular wall preserved systolic contraction and prevented cardiac dysfunction.
Limitations
We used a single dose (5 x 106 cells) for all three cell types. Previous studies have demonstrated that cell dose has a significant influence on the beneficial effects of cell transplantation on ventricular function [24]. It is possible that a better effect could be achieved with a different dose for any of the three muscle cells. To determine the optimal number of cells for transplantation a dose–response curve would need to be done for each cell type. However, it is likely that attempts to enhance cell survival after implantation may be more effective in increasing the number of viable engrafted cells than increasing the number of cells implanted. We have used both vascular endothelial growth factor [27] and insulin-like growth factor [28] and others have used fibroblast growth factor [29] to enhance angiogenesis and cell survival after transplantation.
For clinical application of cell transplantation, cryopreservation may be essential. We have demonstrated previously that cryopreservation reduces the growth characteristics of fetal cardiomyocytes [15]. Therefore, the conditions for cryopreservation may influence the survival after implantation. We were able to achieve a beneficial effect on cardiac function despite cryopreservation of the muscle cells. Further studies will be required to document the influence of cryopreservation with each cell type on the resulting ventricular function.
Conclusion
This study provides preliminary evidence for the benefit of adult muscle cell transplantation. All three muscle cells provided an equivalent response, which lasted longer than the benefit associated with an ACE inhibitor. The mechanism of the beneficial effect of muscle cell transplantation may be an alteration of matrix remodeling because elastin content was higher when function was improved. Finally, this study suggests that cryopreservation may be a practical alternative to facilitate the clinical application of cell transplantation. However, large animal studies will be required to confirm the conclusions reached in these preliminary investigations before clinical trials of alternate cell types.
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Acknowledgments |
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= ACEI; • = control; 
= HC; 
= SKMC; 
= SMC.)